Molecular mechanism of leukocidin GH-integrin CD11b/CD18 recognition and species specificity.

Host-pathogen interactions are central to understanding microbial pathogenesis. The staphylococcal pore-forming cytotoxins hijack important immune molecules but little is known about the underlying molecular mechanisms of cytotoxin-receptor interaction and host specificity. Here we report the structures of a staphylococcal pore-forming cytotoxin, leukocidin GH (LukGH), in complex with its receptor (the α-I domain of complement receptor 3, CD11b-I), both for the human and murine homologs. We observe 2 binding interfaces, on the LukG and the LukH protomers, and show that human CD11b-I induces LukGH oligomerization in solution. LukGH binds murine CD11b-I weakly and is inactive toward murine neutrophils. Using a LukGH variant engineered to bind mouse CD11b-I, we demonstrate that cytolytic activity does not only require binding but also receptor-dependent oligomerization. Our studies provide an unprecedented insight into bicomponent leukocidin-host receptor interaction, enabling the development of antitoxin approaches and improved animal models to explore these approaches.

Randomized, Double-Blind, Placebo-Controlled, Single-Ascending-Dose Study of the Penetration of a Monoclonal Antibody Combination (ASN100) Targeting Staphylococcus aureus Cytotoxins in the Lung Epithelial Lining Fluid of Healthy Volunteers.

ASN100 is a novel antibody combination of two fully human IgG1(κ) monoclonal antibodies (MAbs), ASN-1 and ASN-2, which neutralize six Staphylococcus aureus cytotoxins, alpha-hemolysin (Hla) and five bicomponent leukocidins. We assessed the safety, tolerability, and serum and lung pharmacokinetics of ASN100 in a randomized, double-blind, placebo-controlled single-dose-escalation first-in-human study. Fifty-two healthy volunteers were enrolled and randomized to receive either ASN-1, ASN-2, a combination of both MAbs (ASN100), or a corresponding placebo. Thirty-two subjects in the double-blind dose escalation portion of the study received ASN-1 or ASN-2 at a 200-, 600-, 1,800-, or 4,000-mg dose, or placebo. Eight subjects received both MAbs simultaneously in a 1:1 ratio (ASN100) at 3,600 or 8,000 mg, or they received placebos. Twelve additional subjects received open-label ASN100 at 3,600 or 8,000 mg to assess the pharmacokinetics of ASN-1 and ASN-2 in epithelial lining fluid (ELF) by bronchoalveolar lavage fluid sampling. Subjects were monitored for 98 days (double-blind cohorts) or 30 days (open-label cohorts) for safety assessment. No dose-limiting toxicities were observed, and all adverse events were mild and transient, with only two adverse events considered possibly related to the investigational product. ASN100 exhibited linear serum pharmacokinetics with a half-life of approximately 3 weeks and showed detectable penetration into the ELF. No treatment-emergent anti-drug antibody responses were detected. The toxin neutralizing potency of ASN100 in human serum was confirmed up to 58 days postdosing. The favorable safety profile, ELF penetration, and maintained functional activity in serum supported the further clinical development of ASN100.

Preventing lung pathology and mortality in rabbit Staphylococcus aureus pneumonia models with cytotoxin-neutralizing monoclonal IgGs penetrating the epithelial lining fluid.

Staphylococcus aureus pneumonia is associated with high mortality irrespective of antibiotic susceptibility. Both MRSA and MSSA strains produce powerful cytotoxins: alpha-hemolysin(Hla) and up to five leukocidins - LukSF-PV, HlgAB, HlgCB, LukED and LukGH (LukAB) - to evade host innate defense mechanisms. Neutralizing cytotoxins has been shown to provide survival benefit in rabbit S. aureus pneumonia models. We studied the mechanisms of protection of ASN100, a combination of two human monoclonal antibodies (mAbs), ASN-1 and ASN-2, that together neutralize Hla and the five leukocidins, in rabbit MRSA and MSSA pneumonia models. Upon prophylactic passive immunization, ASN100 displayed dose-dependent increase in survival and was fully protective against all S. aureus strains tested at 5 or 20 mg/kg doses. Macroscopic and microscopic lung pathology, edema rate, and bacterial burden were evaluated 12 hours post infection and reduced by ASN100. Pharmacokinetic analysis of ASN100 in bronchoalveolar-lavage fluid from uninfected animals detected efficient penetration to lung epithelial lining fluid reaching peak levels between 24 and 48 hours post dosing that were comparable to the mAb concentration measured in serum. These data confirm that the ASN100 mAbs neutralize the powerful cytotoxins of S. aureus in the lung and prevent damage to the mucosal barrier and innate immune cells.

Adaptation of the Staphylococcus aureus leukocidin LukGH for the rabbit host by protein engineering.

Host defense against Staphylococcus aureus greatly depends on bacterial clearance by phagocytic cells. LukGH (or LukAB) is the most potent staphylococcal leukocidin towards human phagocytes in vitro, but its role in pathogenesis is obscured by the lack of suitable small animal models because LukGH has limited or no cytotoxicity towards rodent and rabbit compared with human polymorphonuclear cells (PMNs) likely due to an impaired interaction with its cellular receptor, CD11b. We aimed at adapting LukGH for the rabbit host by improving binding to the rabbit homolog of CD11b, specifically its I-domain (CD11b-I). Targeted amino acid substitutions were introduced into the LukH polypeptide to map its receptor interaction site(s). We found that the binding affinity of LukGH variants to the human and rabbit CD11b-I correlated well with their PMN cytotoxicity. Importantly, we identified LukGH variants with significantly improved cytotoxicity towards rabbit PMNs, when expressed recombinantly (10-15-fold) or by engineered S. aureus strains. These findings support the development of small animal models of S. aureus infection with the potential for demonstrating the importance of LukGH in pathogenesis.

Age-related changes in the levels and kinetics of pulmonary cytokine and chemokine responses to Streptococcuspneumoniae in mouse pneumonia models.

Streptococcuspneumoniae is a major human pathogen at the extremes of age. The elderly are particularly vulnerable to S.pneumoniae, the most common causative agent of bacterial pneumonia in this population. Despite the availability of vaccines and antibiotics, mortality rates associated with pneumococcal pneumonia in this age group remain high. In light of globally increasing life-expectancy, a better understanding of the patho-mechanisms of elderly pneumococcal pneumonia, including alterations in innate immune responses, is needed to develop improved therapies. In this study we aimed at investigating how increased susceptibility to pneumococcal infection relates to inflammation kinetics in the aged mouse pneumonia model by determining pulmonary cytokine and chemokine levels and comparing these parameters to those measured in young adult mice. Firstly, we detected overall higher pulmonary cytokine and chemokine levels in aged mice. However, upon induction of pneumococcal pneumonia in aged mice, delayed production of certain analytes, such as IFN-γ, MIG (CXCL9), IP-10 (CXCL10), MCP-1 (CCL2), TARC (CCL17) and MDC (CCL22) became apparent. In addition, aged mice were unable to control excess inflammatory responses: while young mice showed peak inflammatory responses at 20 h and subsequent resolution by 48 h post intranasal challenge, in aged mice increasing cytokine and chemokine levels were measured. These findings highlight the importance of considering multiple time points when delineating inflammatory responses to S.pneumoniae in an age-related context. Finally, correlation between pulmonary bacterial burden and cytokine or chemokine levels in young mice suggested that appropriately controlled inflammatory responses support the host to fight pneumococcal infection.

Assessing the function of pneumococcal neuraminidases NanA, NanB and NanC in in vitro and in vivo lung infection models using monoclonal antibodies.

Streptococcus pneumoniae isolates express up to three neuraminidases (sialidases), NanA, NanB and NanC, all of which cleave the terminal sialic acid of glycan-structures that decorate host cell surfaces. Most research has focused on the role of NanA with limited investigations evaluating the roles of all three neuraminidases in host-pathogen interactions. We generated two highly potent monoclonal antibodies (mAbs), one that blocks the enzymatic activity of NanA and one cross-neutralizing NanB and NanC. Total neuraminidase activity of clinical S. pneumoniae isolates could be inhibited by this mAb combination in enzymatic assays. To detect desialylation of cell surfaces by pneumococcal neuraminidases, primary human tracheal/bronchial mucocilial epithelial tissues were infected with S. pneumoniae and stained with peanut lectin. Simultaneous targeting of the neuraminidases was required to prevent desialylation, suggesting that inhibition of NanA alone is not sufficient to preserve terminal lung glycans. Importantly, we also found that all three neuraminidases increased the interaction of S. pneumoniae with human airway epithelial cells. Lectin-staining of lung tissues of mice pre-treated with mAbs before intranasal challenge with S. pneumoniae confirmed that both anti-NanA and anti-NanBC mAbs were required to effectively block desialylation of the respiratory epithelium in vivo. Despite this, no effect on survival, reduction in pulmonary bacterial load, or significant changes in cytokine responses were observed. This suggests that neuraminidases have no pivotal role in this murine pneumonia model that is induced by high bacterial challenge inocula and does not progress from colonization as it happens in the human host.

Disarming Staphylococcus aureus from destroying human cells by simultaneously neutralizing six cytotoxins with two human monoclonal antibodies.

Pathogenesis of Staphylococcus aureus is increasingly recognized to be driven by powerful toxins. Staphylococcus aureus employs up to six pore-forming toxins to subvert the human host defense and to promote bacterial invasion: alpha-hemolysin that disrupts epithelial and endothelial barriers and five leukocidins that lyse phagocytes involved in bacterial clearance. Previously, we described two human monoclonal antibodies (mAbs), ASN-1 that neutralizes alpha-hemolysin and four leukocidins (LukSF-PV, LukED, HlgAB, HlgCB), and ASN-2 that inactivates the 5th leukocidin, LukGH. In this study we tested the individual and combined effects of ASN-1 and ASN-2 in multiple in vitro models employing relevant human target cells. We found that diverse S. aureus isolates with different genetic backgrounds (based on MLST- and spa-typing) and antibiotic sensitivity (both MRSA and MSSA) displayed greatly different cytotoxin expression patterns influenced by the type of growth medium used. Both mAbs were required to fully prevent the lysis of human neutrophils exposed to the mixture of recombinant cytotoxins or native toxins present in the culture supernatants of S. aureus isolates. Flow cytometry confirmed the protective effects of ASN-1 + ASN-2 (known as ASN100) on granulocytes, monocytes, NK-cells and T-lymphocytes. ASN-1 alone preserved the integrity of a 3D-primary culture of human tracheal/bronchial mucociliary epithelial tissue infected with S. aureus. We conclude that simultaneous inhibition of alpha-hemolysin and five leukocidins by ASN100 blocks cytolytic activity of S. aureus towards human target cells in vitro.

Selective sensitization of human neutrophils to LukGH mediated cytotoxicity by Staphylococcus aureus and IL-8.

OBJECTIVES: Staphylococcus aureus produces up to five bi-component leukocidins - LukSF-PV, gamma-hemolysins AB and CB, LukGH (LukAB) and LukED - to evade innate immunity by lysing phagocytic cells. Species specificity of these leukocidins limits the relevance of animal models, therefore we assessed their individual contribution using human neutrophils. METHODS: Human polymorphonuclear leukocytes (PMNs) were activated with stimuli relevant during bacterial infections and sensitivity to recombinant leukocidins was measured in cell-viability assays. Leukocidin receptor expression was quantified by flow cytometry. RESULTS: We observed greatly variable sensitivities of different PMN preparations towards LukGH. Activation of PMNs by lipopolysaccharide (LPS) or S. aureus culture supernatant (CS) lacking all leukocidins resulted in higher surface expression of CD11b, the LukGH receptor, and greatly enhanced the sensitivity towards LukGH, eliminating the variability observed with unstimulated cells. In contrast, CS induced a decrease in sensitivity of PMNs to the other four leukocidins and reduced surface staining for their cognate receptors (CXCR1, CXCR2, C5aR, C5L2). Delta-toxin and peptidoglycan mimicked the effect of CS. Moreover, IL-8, an important cytokine in neutrophil activation, also selectively increased LukGH sensitivity. Deletion of lukGH, but not other leukocidin genes, prevented PMN killing upon infection with USA300 CA-MRSA. CONCLUSION: Inflammatory signals enhance the susceptibility of human PMNs to lysis by LukGH rendering this toxin dominant among the S. aureus leukocidins in vitro.

Improved Protection in a Rabbit Model of Community-Associated Methicillin-Resistant Staphylococcus aureus Necrotizing Pneumonia upon Neutralization of Leukocidins in Addition to Alpha-Hemolysin.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), especially the USA300 pulsotype, is a frequent cause of skin and soft tissue infections and severe pneumonia. Despite appropriate antibiotic treatment, complications are common and pneumonia is associated with high mortality. S. aureus strains express multiple cytotoxins, including alpha-hemolysin (Hla) and up to five bicomponent leukocidins that specifically target phagocytic cells for lysis. CA-MRSA USA300 strains carry the genes for all six cytotoxins. Species specificity of the leukocidins greatly contributes to the ambiguity regarding their role in S. aureus pathogenesis. We performed a comparative analysis of the leukocidin susceptibility of human, rabbit, and mouse polymorphonuclear leukocytes (PMNs) to assess the translational value of mouse and rabbit S. aureus models. We found that mouse PMNs were largely resistant to LukSF-PV, HlgAB, and HlgCB and susceptible only to LukED, whereas rabbit and human PMNs were highly sensitive to all these cytotoxins. In the rabbit pneumonia model with a USA300 CA-MRSA strain, passive immunization with a previously identified human monoclonal antibody (MAb), Hla-F#5, which cross-neutralizes Hla, LukSF-PV, HlgAB, HlgCB, and LukED, provided full protection, whereas an Hla-specific MAb was only partially protective. In the mouse USA300 CA-MRSA pneumonia model, both types of antibodies demonstrated full protection, suggesting that Hla, but not leukocidin(s), is the principal virulence determinant in mice. As the rabbit recapitulates the high susceptibility to leukocidins characteristic of humans, this species represents a valuable model for assessing novel, cytotoxin-targeting anti-S. aureus therapeutic approaches.

Context matters: The importance of dimerization-induced conformation of the LukGH leukocidin of Staphylococcus aureus for the generation of neutralizing antibodies.

LukGH (LukAB) is a potent leukocidin of Staphylococcus aureus that lyses human phagocytic cells and is thought to contribute to immune evasion. Unlike the other bi-component leukocidins of S. aureus, LukGH forms a heterodimer before binding to its receptor, CD11b expressed on professional phagocytic cells, and displays significant sequence variation. We employed a high diversity human IgG1 library presented on yeast cells to discover monoclonal antibodies (mAbs) neutralizing the cytolytic activity of LukGH. Recombinant LukG and LukH monomers or a LukGH dimer were used as capture antigens in the library selections. We found that mAbs identified with LukG or LukH as bait had no or very low toxin neutralization potency. In contrast, LukGH dimer-selected antibodies proved to be highly potent, and several mAbs were able to neutralize even the most divergent LukGH variants. Based on biolayer interferometry and mesoscale discovery, the high affinity antibody binding site on the LukGH complex was absent on the individual monomers, suggesting that it was generated upon formation of the LukG-LukH dimer. X-ray crystallography analysis of the complex between the LukGH dimer and the antigen-binding fragment of a very potent mAb (PDB code 5K59) indicated that the epitope is located in the predicted cell binding region (rim domain) of LukGH. The corresponding IgG inhibited the binding of LukGH dimer to target cells. Our data suggest that knowledge of the native conformation of target molecules is essential to generate high affinity and functional mAbs.

Membrane Anchors of the Structural Flavivirus Proteins and Their Role in Virus Assembly.

UNLABELLED: The structural proteins of flaviviruses carry a unique set of transmembrane domains (TMDs) at their C termini that are derived from the mode of viral polyprotein processing. They function as internal signal and stop-transfer sequences during protein translation, but possible additional roles in protein interactions required during assembly and maturation of viral particles are ill defined. To shed light on the role of TMDs in these processes, we engineered a set of tick-borne encephalitis virus mutants in which these structural elements were replaced in different combinations by the homologous sequences of a distantly related flavivirus (Japanese encephalitis virus). The effects of these modifications were analyzed with respect to protein synthesis, viral particle secretion, specific infectivity, and acidic-pH-induced maturation processes. We provide evidence that interactions involving the double-membrane anchor of the envelope protein E (a unique feature compared to other viral fusion proteins) contribute substantially to particle assembly, stability, and maturation. Disturbances of the inter- and intra-TMD interactions of E resulted in the secretion of a larger proportion of capsidless subviral particles at the expense of whole virions, suggesting a possible role in the still incompletely understood mechanism of capsid integration during virus budding. In contrast, the TMD initially anchoring the C protein to the endoplasmic reticulum membrane does not appear to take part in envelope protein interactions. We also show that E TMDs are involved in the envelope protein rearrangements that are triggered by acidic pH in the trans-Golgi network and represent a hallmark of virus maturation. IMPORTANCE: The assembly of flaviviruses occurs in the endoplasmic reticulum and leads to the formation of immature, noninfectious particles composed of an RNA-containing capsid surrounded by a lipid membrane, with the two integrated envelope proteins, prM and E, arranged in an icosahedral lattice. The mechanism by which the capsid is formed and integrated into the budding viral envelope is currently unknown. We provide evidence that the transmembrane domains (TMDs) of E are essential for the formation of capsid-containing particles and that disturbances of these interactions lead to the preferential formation of capsidless subviral particles at the expense of whole virions. E TMD interactions also appear to be essential for the envelope protein rearrangements required for virus maturation and for the generation of infectious virions. Our data thus provide new insights into the biological functions of E TMDs and extend their role during viral polyprotein processing to additional functions in particle assembly and maturation.

Structure-function analysis of heterodimer formation, oligomerization, and receptor binding of the Staphylococcus aureus bi-component toxin LukGH.

The bi-component leukocidins of Staphylococcus aureus are important virulence factors that lyse human phagocytic cells and contribute to immune evasion. The γ-hemolysins (HlgAB and HlgCB) and Panton-Valentine leukocidin (PVL or LukSF) were shown to assemble from soluble subunits into membrane-bound oligomers on the surface of target cells, creating barrel-like pore structures that lead to cell lysis. LukGH is the most distantly related member of this toxin family, sharing only 30-40% amino acid sequence identity with the others. We observed that, unlike other leukocidin subunits, recombinant LukH and LukG had low solubility and were unable to bind to target cells, unless both components were present. Using biolayer interferometry and intrinsic tryptophan fluorescence we detected binding of LukH to LukG in solution with an affinity in the low nanomolar range and dynamic light scattering measurements confirmed formation of a heterodimer. We elucidated the structure of LukGH by x-ray crystallography at 2.8-Šresolution. This revealed an octameric structure that strongly resembles that reported for HlgAB, but with important structural differences. Structure guided mutagenesis studies demonstrated that three salt bridges, not found in other bi-component leukocidins, are essential for dimer formation in solution and receptor binding. We detected weak binding of LukH, but not LukG, to the cellular receptor CD11b by biolayer interferometry, suggesting that in common with other members of this toxin family, the S-component has the primary contact role with the receptor. These new insights provide the basis for novel strategies to counteract this powerful toxin and Staphylococcus aureus pathogenesis.

Five birds, one stone: neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody.

Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.

α-Hemolysin activity of methicillin-susceptible Staphylococcus aureus predicts ventilator-associated pneumonia.

RATIONALE: Colonization of lower airways by Staphylococcus aureus is a risk factor for the development of ventilator-associated tracheobronchitis (VAT) and ventilator-associated pneumonia (VAP). However, little is known about the virulence factors of methicillin-sensitive and -resistant S. aureus (MSSA and MRSA) that may influence host colonization and progression to VAT and VAP. OBJECTIVES: We evaluated MRSA and MSSA endotracheal aspirates (ETA) for genotype and α-hemolysin activity in relation to the development of VAT and VAP. METHODS: Serial S. aureus ETA isolates from ventilated patients were analyzed for methicillin resistance, molecular type by Multi-Locus Sequence Typing and spa-typing, and α-hemolysin activity by semiquantitative analysis of hemolysis on sheep blood agar and quantitative measurement of cytolysis of human lung epithelial cells. The virulence of selected strains was assessed in mice by intranasal challenge. MEASUREMENTS AND MAIN RESULTS: We detected S. aureus from ETA samples in a quarter of the 231 ventilated patients analyzed; one-third of them developed VAP. VAP patients (n = 15) were mainly infected by MSSA strains (87%), whereas colonized individuals (n = 18) not progressing to disease mainly carried MRSA strains (68%). MSSA isolates from colonized or VAT patients exhibited significantly lower α-hemolysin activity than those from VAP cases; however, no such relationship was found with MRSA strains. α-Hemolysin activity of S. aureus isolates was predictive for virulence in mouse pneumonia model. CONCLUSIONS: MSSA strains with strong blood agar hemolysis and high α-hemolysin activity are markers for VAP, but not VAT, and might be considered in differential diagnosis and initiation of therapy.

Mutational analysis of three predicted 5'-proximal stem-loop structures in the genome of tick-borne encephalitis virus indicates different roles in RNA replication and translation.

Flavivirus gene expression is modulated by RNA secondary structure elements at the terminal ends of the viral RNA molecule. For tick-borne encephalitis virus (TBEV), four stem-loop (SL) elements have been predicted in the first 180 nucleotides of the viral genome: 5'-SL1, 5'-SL2, 5'-SL3 and 5'-SL4. The last three of these appear to be unique to tick-borne flaviviruses. Here, we report their characterization by mutagenesis in a TBEV luciferase reporter system. By manipulating their thermodynamic properties, we found that an optimal stability of the 5'-SL2 is required for efficient RNA replication. 5'-SL3 formation is also important for viral RNA replication, but although it contains the viral start codon, its formation is dispensable for RNA translation. 5'-SL4 appears to facilitate both RNA translation and replication. Our data suggest that maintenance of the balanced thermodynamic stability of these SL elements is important for temporal regulation of its different functions.

Functional microRNA generated from a cytoplasmic RNA virus.

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that play a pivotal role in the regulation of posttranscriptional gene expression in a wide range of eukaryotic organisms. Although DNA viruses have been shown to encode miRNAs and exploit the cellular RNA silencing machinery as a convenient way to regulate viral and host gene expression, it is generally believed that this pathway is not available to RNA viruses that replicate in the cytoplasm of the cell because miRNA biogenesis is initiated in the nucleus. In fact, among the >200 viral miRNAs that have been experimentally verified so far, none is derived from an RNA virus. Here, we show that a cytoplasmic RNA virus can indeed encode and produce a functional miRNA. We introduced a heterologous miRNA-precursor stem-loop sequence element into the RNA genome of the flavivirus tick-borne encephalitis virus, and this led to the production of a functional miRNA during viral infection without impairing viral RNA replication. These findings demonstrate that miRNA biogenesis can be used by cytoplasmic RNA viruses to produce regulatory molecules for the modulation of the transcriptome.

Analysis of the effects of alterations in the tick-borne encephalitis virus 3'-noncoding region on translation and RNA replication using reporter replicons.

The 3'-noncoding region (3'-NCR) of the flavivirus genome includes a variable region that tolerates the insertion of heterologous genetic information. Natural isolates of tick-borne encephalitis virus (TBEV) have particularly long variable regions, which, for some strains, include an internal poly(A) tract. We constructed luciferase reporter replicons of TBEV to analyze the impact of various manipulations of the 3'-NCR on viral RNA translation and replication. The choice of the reporter gene, its position and processing within the viral polyprotein, and the choice of standards were found to be important for obtaining a sensitive and reliable test system. We observed that truncation or complete removal of the internal poly(A) tract, or even the entire variable region, had no significant impact on translation and replication of the RNA in mammalian cell culture. Substitution of the variable region with foreign genetic elements impaired RNA replication to various degrees but generally had no influence on viral translation. Expression cassettes driven by an IRES element inhibited RNA replication more strongly than did repetitive protein-binding elements derived from a bacteriophage, even when the ligand that binds these elements was co-expressed in the cells. Previously identified mutations in the IRES partially relieved this inhibition when introduced into the reporter replicon but provided no evidence for intramolecular competition for translation factors. Impairment of replication appeared to depend more on the type of foreign insert than on its length. These results provide a rational basis for the construction of TBEV-based vectors or vaccines as well as molecular tools for studying flavivirus replication.